Dynamic roles of angiopoietin-like proteins 1, 2, 3, 4, 6 and 7 in the survival and enhancement of ex vivo expansion of bone-marrow hematopoietic stem cells

Recent advances in hematopoietic stem cells(HSCs)expansion by growth factors including angiopoietin-like proteins(Angptls)have opened up the possibility to use HSCs in regenerative medicine.However,the unavailability of true in vitro HSCs expansion by these growth factors has limited the understanding of the cellular and molecular mechanism of HSCs expansion.Here,we report the functional role of mouse Angptls 1,2,3,4,6 and 7 and growth factors SCF,TPO,IGF-2 and FGF-1 on purified mouse bone-marrow(BM)Lineage-Sca-1+(Lin-Sca-1+)HSCs.The recombinant retroviral transduced-CHO-S cells that secrete Angptls in serum-free medium were used alone or in combination with growth factors(SCF,TPO,IGF-2 and FGF-1).None of the Angptls stimu-lated HSC proliferation,enhanced or inhibited HSCs colony formation,but they did support the survival of HSCs.By contrast,any of the six Angptls together with saturating levels of growth factors dramatically stimulated a 3-to 4.5-fold net expansion of HSCs compared to stimulation with a combination of those growth factors alone.These findings lead to an understanding of the basic function of Angptls on signaling pathways for the survival as well as expansion of HSCs in the bone marrow niche.

Ex vivo expansion of regulatory T cells for clinical applications against graft-versus-host disease in allogeneic hematopoietic stem cell transplantation

Objective To review the characteristics of regulatory T cells （Tregs） and ex vivo expansion of Tregs for treatment of graftversus-host disease （GVHD）.Data sources The data used in this review were retrieved from PubMed （1970-2013）.The terms ＂ex vivo expansion＂,＂regulatory T cell＂,and ＂graft-versus-host disease＂ were used for literature search.Study selection The publications about the characteristics of Tregs,ex vivo expansion of Tregs and clinical applications of Tregs against GVHD were identified,retrieved and reviewed.Results Tregs can be classified as natural Tregs （nTregs） and induced Tregs （iTregs）.Both subsets share most Treg features.Given their immunosuppressive property,Tregs have been tested for their capability of preventing GVHD.The bottleneck of Treg therapy is the limited numbers of naturally existing Tregs.To solve this problem,ex vivo expansion of nTregs or iTregs has been executed.The initial data indicate Treg therapy is effective in reducing GVHD without compromising graft-versus-leukemia （GVL）.Conclusion Ex vivo expansion of Tregs is a reliable way to prepare sufficient number of Tregs for management of GVHD.

Successful ex vivo expansion of mouse hematopoietic stem cells

Ex vivo expansion of hematopoietic stem cells(HSCs)is considered the holy grail in stem cell biology and therapy,as it has long been difficult to make this procedure possible.Yamazaki’s research team has established new,polyvinyl alcohol-based culture conditions and shown a significant expansion of mouse HSCs from a small number of cells after a month of culture.Surprisingly,expanded HSCs were able to reconstitute unconditioned normal mice.There is generally a technical concern in limiting dilution assay to estimate a fold-expansion of HSCs.But,this work paves the way toward expansion of human HSCs useful for transplantation medicine.

The Cross-talk between ROS and p38MAPKα in the Ex Vivo Expanded Human Umbilical Cord Blood CD133^+ Cells

This study investigated the correlation between and compared the effects of reactive oxygen species（ROS） and p38 mitogen-activated protein kinase α（p38MAPKα） in the ex vivo expanded umbilical cord blood（hUCB） CD133＋ cells.hUCB CD133＋ cells were cultured in the hematopoietic stem cells（HSCs） culture medium with N-acetylcysteine（NAC,an anti-oxidant）,p38MAPKα-specific inhibitor（SB203580） or their combination.The levels of ROS and expression of phosphorylated p38MAPKα（p-p38） in CD133＋ cells were flow cytometrically detected.The efficacy of ex vivo expansion was evaluated by the density of CD133＋ cell sub-group colony-forming cells（CFC） and cobblestone area-forming cells（CAFC） assay.Our results showed decreased ROS levels in NAC,SB203580,and their combination treatment groups were almost 37%,48%,and 85%,respectively.Furthermore,SB203580 abrogated the activation of p38MAPKα more obviously than NAC.Moreover,the CD133＋ cells in SB203580 treatment group had a 21.93±1.36-fold increase,and 14.50±1.19-fold increase in NAC treatment group,but only 10.13±0.57-fold increase in control group.In addition,SB203580 treatment led a higher level increase in the number of CFU and CAFC than NAC did.These findings suggested that,in expanded CD133＋ cells,ROS activates p38MAPKα,which,in turn,induces ROS production,and p38MAPKα might be the most suitable regulator in ROS-p38MAPKα pathway for the promotion of HSCs ex vivo expansion.

Effect of Angiotensin Ⅱ on Cord Blood CD^(34+) Cells Expansion in vitro

In order to investigate the influence of angiotensin Ⅱ on hematopoietic system, CD34 + cells in cord blood were purified, and the effects of angiotensin Ⅱ in combination with various cytokines on their growth and differentiation were studied by cell culture in vitro. It was found that angiotensin Ⅱ in suspending medium could stimulate both BFU-E and CFU-GM expansion. The number of BFU-E and CFU-GM was increased with the increases of angiotensin Ⅱ concentrations during a certain range. In addition, the expansion fold of CFU-GM was increased from 2.3±0.8 times to 7.8±2.3 times when angiotensin Ⅱ was added in the presence of SCF+G-CSF+GM-CSF+IL-3 cytokines mixture. Similarly, the expansion fold of BFU-E was increased from 3.1±1.8 times to 9 2±2.3 times with angiotensin Ⅱ in the presence of SCF+EPO+TPO+IL-3. In the semi-solid medium, angiotensin Ⅱ could stimulate CFU-GM expansion but had no effect on the growth of BFU-E. In conclusion, angiotensin Ⅱ had some stimulating effects on cord blood hematopoietic progenitors expansion in vitro in the presence of other cytokines.

Dendritic cell-mimicking scaffolds for ex vivo T cell expansion

We propose an ex vivo T cell expansion system that mimics natural antigen-presenting cells(APCs)for adoptive cell therapy(ACT).Microfiber scaffolds coated with dendritic cell(DC)membrane replicate physicochemical properties of dendritic cells specific for T cell activation such as rapid recognition by T cells,long duration of T cell tethering,and DC-specific co-stimulatory cues.The DC membrane-coated scaffold is first surface-immobilized with T cell stimulatory ligands,anti-CD3(αCD3)and anti-CD28(αCD28)antibodies,followed by adsorption of releasable interleukin-2(IL-2).The scaffolds present both surface and soluble cues to T cells ex vivo in the same way that these cues are presented by natural APCs in vivo.We demonstrate that the DC-mimicking scaffold promotes greater polyclonal expansion of primary human T cells as compared toαCD3/αCD28-func-tionalized Dynabead.More importantly,major histocompatibility complex molecules derived from the DC membrane of the scaffold allow antigen-specific T cell expansion with target cell-specific killing ability.In addition,most of the expanded T cells(~97%)can be harvested from the scaffold by density gradient centri-fugation.Overall,the DC-mimicking scaffold offers a scalable,modular,and customizable platform for rapid expansion of highly functional T cells for ACT.

Expansion of CD34^+ cells from human umbilical cord blood by FL and/or TPO gene transfected human marrow stromal cell lines

To elucidate the effect of gene transfected marrow stromal cell on expansion of human cord blood CD34+ cells, a culture system was established in which FL and TPO genes were transfected into human stromal cell line HFCL. To establish gene transfected stromal cells co-culture system, cord blood CD34+ cells were purified by using a magnetic beads sorting system. The number of all cells and the number of CD34+ cells and CFC (CFU-GM and BFU-E) were counted in different culture systems. The results showed that in all 8 culture systems, SCF+IL-3+HFT manifested the most potent combination, with the number of total nucleated cells increasing by (893.3±52.1)-fold, total progenitor cells (CFC) by (74.5±5.2)-fold and CD34+ cells by 15.7-fold.Maximal expansions of CFC and CD34+ cells were observed at the end of the second week of culture. Within 14 days of culture, (78.1 ± 5.5)-fold and (57.0 ± 19.7)-fold increases in CFU-GM and BFU-E were obtained. Moreover, generation of LTC-IC from amplified CD34+ cells within 28 days was found only in two combinations, I.e. SCF+IL-3+FL+TPO and SCF+IL-3+HFT, and there was no significant difference between these two groups statistically. These results suggest that human umbilical cord blood CD34+ cells can be extensively expanded ex vivo by using gene transfected stromal cells along with cytokines.

Experimental study on ex vivo expanded hematopoietic stem/progenitor in the two step culture from human umbilical cord blood transplanted into NOD/SCID mice

The effects of hematopoietic stem/progenitor cells(HSPCs)expanded in the two step coculture with human bone marrow mesenchymal stem cells(hMSCs)on the hematopoietic reconstruction of irradiated NOD/SCID mice were studied.Mononuclear cells(MNCs)were isolated from human umbilical cord blood(UCB)and cultured in the non-coculture scheme of rhSCF+rhG−CSF+rhMDGF combination and the coculture scheme of rhSCF+rhG−CSF+rhMDGF+hMSCs.Sublethally-irradiated NOD/SCID mice were transplanted with ex vivo expanded HSPCs with the dose of 8.5×10^(6) cells per mouse.After transplantation,the dynamics of WBC in the transplanted mice was measured periodically,and the Alu sequence fragment special for human in the transplanted mice was inspected by PCR.Results showed that the coculture scheme increased proliferation of UCB-derived HSPCs.After transplantation with expanded HSPCs,the population of WBC in the transplanted mice increased in 12 d and reached the first peak in 25 d,then showed the second increasing of WBC in 45~55 d.Expanded cells from the coculture scheme appeared to be favorable for the second increasing of WBC in the transplanted mice.After 85 d,the Alu sequence fragment was detected in the probability of 87.5%(7/8)for the non-coculture scheme and 88.9%(8/9)for the coculture scheme.

Clinical Grade of Gerneration of Dendritic Cells for Immunotherapy

In order to develop a protocol for clinical grade generation of dendritic cells (DCs) for cancer immumotherapy, aphereses were performed with the continuous flow cell separator and mate- rials were derived from 10 leukemia patients that had achieved complete remission. Peripheral blood monocytes were cultured in vitro with GM-CSF, IL-4 for 6 days, then TNF-α (the TNF-α group) or TNF-α, IL-1β, IL-6, PGE2 (the cytokine mixture group) were added to promote maturation. Cell number was counted by hematology analyzer, and phenotype study (CD1a, CD14, CD83) was carried out by flow cytometry, and the function of DCs was examined by mixed lymphocyte reaction. The results showed that (0.70±0.13)×107/mL (the TNF-α group) and (0.79±0.04)×107/mL (the cytokine mixture group) DCs were generated respectively in peripheral blood obtained by leucapheresis. The phenotypes were as follows: CD1a+ (74.65±4.45)%, CD83+ (39.50±4.16)%, CD14+ (2.90±1.76)% in TNF-α group, and CD1a+ (81.86±5.87)%, CD83+ (81.65±6.36)%, CD14+ (2.46±1.68)% in the cyto- kine mixture group. It was concluded that leucapheresis may be a feasible way to provide large num- ber of peripheral blood monocytes for DC generation, and combined administration of TNF-α, IL-1β, IL-6, and PGE2 may greatly promote maturity.

Interleukin-12 supports in vitro self-renewal of long-term hematopoietic stem cells

Hematopoietic stem cells(HSCs)self-renew or differentiate through division.Cytokines are essential for inducing HSC division,but the optimal cytokine combination to control self-renewal of HSC in vitro remains unclear.In this study,we compared the effects of interleukin-12(IL-12)and thrombopoietin(TPO)in combination with stem cell factor(SCF)on in vitro self-renewal of HSCs.Single-cell assays were used to overcome the heterogeneity issue of HSCs,and serum-free conditions were newly established to permit reproduction of data.In single-cell cultures,CD150^(+)CD48^(-)CD41^(-)CD34^(-)c-Kit^(+)Sca-1^(+)lineage^(-)SCs divided significantly more slowly in the presence of SCF+IL-12 compared with cells in the presence of SCF+TPO.Serial transplantation of cells from bulk and clonal cultures revealed that TPO was more effective than IL-12 at supporting in vitro self-renewal of short-term(<6 months)HSCs,resulting in a monophasic reconstitution wave formation,whereas IL-12 was more effective than TPO at supporting the in vitro selfrenewal of long-term(>6 months)HSCs,resulting in a biphasic reconstitution wave formation.The control of division rate in HSCs appeared to be crucial for preventing the loss of self-renewal potential from their in vitro culture.